Thioflavin derivatives for use in antemortem diagnosis of Alzheimer&#39;s disease and in vivo imaging and prevention of amyloid deposition

ABSTRACT

This invention relates to novel thioflavin derivatives, methods of using the derivatives in, for example, in vivo imaging of patients having neuritic plaques, pharmaceutical compositions comprising the thioflavin derivatives and method of synthesizing the compounds. The compounds find particular use in the diagnosis and treatment of patients having diseases where accumulation of neuritic plaques are prevalent. The disease states or maladies include but are not limited to Alzheimer&#39;s disease, familial Alzheimer&#39;s disease, Down&#39;s Syndrome and homozygotes for the apolipoprotein E4 allele.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.09/935,767, filed Aug. 24, 2001, now abandoned, which is aNon-Provisional of U.S. Patent Application 60/227,601, filed Aug. 24,2000, incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to specific thioflavin derivatives thatare suitable for imaging amyloid deposits in living patients. Morespecifically, the present invention relates to a method of imagingamyloid deposits in brain in vivo to allow antemortem diagnosis ofAlzheimer's disease with thioflavin derivatives. The present inventionalso relates to therapeutic uses for such compounds.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (“AD”) is a neurodegenerative illness characterizedby memory loss and other cognitive deficits. McKhann et al., Neurology34: 939 (1984). It is the most common cause of dementia in the UnitedStates. AD can strike persons as young as 40-50 years of age, yet,because the presence of the disease is difficult to determine withoutdangerous brain biopsy, the time of onset is unknown. The prevalence ofAD increases with age, with estimates of the affected populationreaching as high as 40-50% by ages 85-90. Evans et al., JAMA 262: 2551(1989); Katzman, Neurology 43: 13 (1993).

In practice, AD is definitively diagnosed through examination of braintissue, usually at autopsy. Khachaturian, Arch. Neurol. 42: 1097 (1985);McKhann et al., Neurology 34: 939 (1984). Neuropathologically, thisdisease is characterized by the presence of neuritic plaques (NP),neurofibrillary tangles (NFT), and neuronal loss, along with a varietyof other findings. Mann, Mech. Ageing Dev. 31: 213 (1985). Post-mortemslices of brain tissue of victims of Alzheimer's disease exhibit thepresence of amyloid in the form of proteinaceous extracellular cores ofthe neuritic plaques that are characteristic of AD.

The amyloid cores of these neuritic plaques are composed of a proteincalled the β-amyloid (Aβ) that is arranged in a predominatelybeta-pleated sheet configuration. Mori et al., Journal of BiologicalChemistry 267: 17082 (1992); Kirschner et al., PNAS 83: 503 (1986).Neuritic plaques are an early and invariant aspect of the disease. Mannet al., J. Neurol. Sci. 89: 169; Mann, Mech. Ageing Dev. 31: 213 (1985);Terry et al., J. Neuropathol. Exp. Neurol 46: 262 (1987).

The initial deposition of Aβ probably occurs long before clinicalsymptoms are noticeable. The currently recommended “minimum microscopiccriteria” for the diagnosis of AD is based on the number of neuriticplaques found in brain. Khachaturian, Arch. Neurol., supra (1985).Unfortunately, assessment of neuritic plaque counts must be delayeduntil after death.

Amyloid-containing neuritic plaques are a prominent feature of selectiveareas of the brain in AD as well as Down's Syndrome and in personshomozygous for the apolipoprotein E4 allele who are very likely todevelop AD. Corder et al., Science 261: 921 (1993); Divry, P., J.Neurol. Psych. 27: 643-657 (1927); Wisniewski et al., in Zimmerman, H.M. (ed.): PROGRESS IN NEUROPATHOLOGY (Grune and Stratton, N.Y. 1973) pp.1-26.

Brain amyloid is readily demonstrated by staining brain sections withthioflavin S or Congo red. Puchtler et al., J. Histochem. Cytochem. 10:35 (1962). Congo red stained amyloid is characterized by a dichroicappearance, exhibiting a yellow-green polarization color. The dichroicbinding is the result of the beta-pleated sheet structure of the amyloidproteins. Glenner, G. N. Eng. J. Med. 302: 1283 (1980). A detaileddiscussion of the biochemistry and histochemistry of amyloid can befound in Glenner, N. Eng. J. Med., 302: 1333 (1980).

Thus far, diagnosis of AD has been achieved mostly through clinicalcriteria evaluation, brain biopsies and post-mortem tissue studies.Research efforts to develop methods for diagnosing Alzheimer's diseasein vivo include (1) genetic testing, (2) immunoassay methods and (3)imaging techniques.

Evidence that abnormalities in Aβ metabolism are necessary andsufficient for the development of AD is based on the discovery of pointmutations in the Aβ precursor protein in several rare families with anautosomal dominant form of AD. Hardy, Nature Genetics 1: 233 (1992);Hardy et al., Science 256: 184 (1992). These mutations occur near the N-and C-terminal cleavage points necessary for the generation of Aβ fromits precursor protein. St. George-Hyslop et al., Science 235: 885(1987); Kang et al., Nature 325: 733 (1987); Potter WO 92/17152. Geneticanalysis of a large number of AD families has demonstrated, however,that AD is genetically heterogeneous. St. George-Hyslop et al., Nature347: 194 (1990). Linkage to chromosome 21 markers is shown in only somefamilies with early-onset AD and in no families with late-onset AD. Morerecently a gene on chromosome 14 whose product is predicted to containmultiple transmembrane domains and resembles an integral membraneprotein has been identified by Sherrington et al., Nature 375: 754-760(1995). This gene may account for up to 70% of early-onset autosomaldominant AD. Preliminary data suggests that this chromosome 14 mutationcauses an increase in the production of Aβ. Scheuner et al., Soc.Neurosci. Abstr. 21: 1500 (1995). A mutation on a very similar gene hasbeen identified on chromosome 1 in Volga German kindreds withearly-onset AD. Levy-Lahad et al., Science 269: 973-977 (1995).

Screening for apolipoprotein E genotype has been suggested as an aid inthe diagnosis of AD. Scott, Nature 366: 502 (1993); Roses, Ann. Neurol.38: 6-14 (1995). Difficulties arise with this technology, however,because the apolipoprotein E4 allele is only a risk factor for AD, not adisease marker. It is absent in many AD patients and present in manynon-demented elderly people. Bird, Ann. Neurol. 38: 2-4 (1995).

Immunoassay methods have been developed for detecting the presence ofneurochemical markers in AD patients and to detect an AD related amyloidprotein in cerebral spinal fluid. Warner, Anal. Chem. 59: 1203A (1987);World Patent No. 92/17152 by Potter; Glenner et al., U.S. Pat. No.4,666,829. These methods for diagnosing AD have not been proven todetect AD in all patients, particularly at early stages of the diseaseand are relatively invasive, requiring a spinal tap. Also, attempts havebeen made to develop monoclonal antibodies as probes for imaging of Aβ.Majocha et al., J. Nucl. Med., 33: 2184 (1992); Majocha et al., WO89/06242 and Majocha et al., U.S. Pat. No. 5,231,000. The majordisadvantage of antibody probes is the difficulty in getting these largemolecules across the blood-brain barrier. Using antibodies for in vivodiagnosis of AD would require marked abnormalities in the blood-brainbarrier in order to gain access into the brain. There is no convincingfunctional evidence that abnormalities in the blood-brain barrierreliably exist in AD. Kalaria, Cerebrovascular & Brain MetabolismReviews 4: 226 (1992).

Radiolabeled Aβ peptide has been used to label diffuse, compact andneuritic type plaques in sections of AD brain. See Maggio et al., WO93/04194. However, these peptides share all of the disadvantages ofantibodies. Specifically, peptides do not normally cross the blood-brainbarrier in amounts necessary for imaging and because these probes reactwith diffuse plaques, they may not be specific for AD.

Neuritic plaques and neurofibrillary tangles are the two mostcharacteristic pathological hallmarks of AD. Klunk and Abraham,Psychiatric Development, 6:121-152 (1988). Plaques occur earliest inneocortex where they are relatively evenly distributed. Thal et al.,Neurology 58:1791-1800 (2002). Tangles appear first in limbic areas suchas the transentorhinal cortex and progress in a predictable topographicpattern to the neocortex. Braak and Braak, Acta Neuropathologica82:239-259 (1991). Arnold et al. mapped the distribution of NFT andneuritic plaques in the brains of patients with AD. Arnold et al.,Cereb. Cortex 1:103-116 (1991). Compared to NFT, neuritic plaques were,in general, more evenly distributed throughout the cortex, with theexceptions of notably fewer neuritic plaques in limbic periallocortexand allocortex (the areas with greatest NFT density). By thioflavin-Sstaining, temporal and occipital lobes had the highest neuritic plaquedensities, limbic and frontal lobes had the lowest, and parietal lobewas intermediate. Arriagada et al., Neurology 42:1681-1688 (1992).Arriagada et al. studied the topographic distribution of AD-typepathologic changes in the brains of presumed nondemented elderlyindividuals. Their observations suggest that most individuals over theage of 55 have at least a few NFT and plaques. Immunohistochemicallydefined subtypes of SP had distinct patterns of distribution withAβ-immunoreactive plaques present in neocortical areas much greater thanlimbic areas and Alz-50 immunoreactive plaques being infrequent andlimited to those areas that contain Alz-50-positive neurons and NFT.These patterns suggested a commonality in the pathologic processes thatlead to NFT and SP in both aging and AD.

There remains debate as to whether plaques and tangles are byproducts ofthe neurodegenerative process found in AD or whether they are the causeof neuronal cell death. Ross, Current Opinion in Neurobiol. 96:644-650(1996); Terry, J. of Neuropath. & Exp. Neurol. 55:1023-1025 (1996);Terry, J Neural Transmission—Suppl. 53:141-145 (1998). Evidence is clearthat neocortical and hippocampal synapse loss correlates well withpre-morbid cognitive status. Some researchers suggest that disruption ofmicrotubule structure and function, caused by the hyperphosphorylationof the microtubule-associated protein, tau, plays the key etiologic rolein synapse loss in particular and AD in general. Terry, J. of Neuropath.& Exp. Neurol. 55:1023-1025 (1996); Terry, J of NeuralTransmission—Suppl. 53:141-145 (1998). Oxidative damage and membranebreakdown have been proposed to play important roles in AD. Perry, FreeRadical Biology & Medicine 28:831-834 (2000); Pettegrew et al., Annalsof the New York Academy of Sciences 826:282-306 (1997). Vascular factorsincluding subtle, chronic cerebral hypoperfusion also have beenimplicated in the pathogenesis of AD. De la Torre, Annals of the NewYork Academy of Sciences 903:424-436 (2000); Di Iorio et al., Aging(Milano) 11:345-352 (1999). While all of these factors are likely toplay some role in the pathogenesis of AD, increasing evidence points toabnormalities in the processing of the amyloid-beta (Aβ) peptide, a 4 kDpeptide that aggregates into a fibrillar, β-pleated sheet structure.Glenner and Wong, Biochemical & Biophysical Research Communications120:885-890 (1984). Aβ has been proposed to play an important role inthe pathogenesis of AD for several reasons: 1) Aβ deposits are theearliest neuropathological markers of AD in Down's Syndrome, and canprecede NFT formation by several decades Mann et al., Neurodegeneration1:201-215 (1992); Naslund, et al., JAMA 283:1571-1577 (2000). 2)β-amyloidosis is relatively specific to AD and closely relateddisorders; Selkoe, Trends in Neurosciences 16:403-409 (1993); 3) Aβ istoxic to cultured neurons, Yankner Neurobiol. Aging 13:615-616 (1992);Mattson et al., J. Neuroscience 12:376-389 (1992); Shearman et al.,Proc. Natl. Acad. Sci. USA 91:1470-1474 (1994), a toxicity that appearsto be dependent on β-sheet secondary structure and aggregation into atleast oligomers. Lambert et al. Proc. Natl. Acad. Sci. USA 95:6448-6453(1989); Pike et al., J. Neuroscience 13:1676-1687 (1993); Simmons etal., Molecular Pharmacology 45:373-379 (1994). Although Aβ surely existsin an equilibrium distributed across monomeric, oligomeric andfibrillar/plaque fractions, the oligomeric form of Aβ has been stronglyimplicated as the key neurotoxic component. Selkoe, Alzheimer disease,edited by R. D. Terry, et al., pp. 293-310 Lippincott Williams andWilkins, Philadelphia (1999); Selkoe, Science 298, 789-91 (2002).Recognition of the toxic effects of oligomeric Aβ has formed a basis forcompromise for some opponents of the “amyloid cascade hypothesis” of AD.Terry, Ann. Neurol. 49:684 (2001). Perhaps the strongest evidence for arole of Aβ in the pathogenesis of AD comes from the finding of mutationsin the amyloid precursor protein (APP) gene which lead to some forms ofearly onset familial AD. Goate et al., Nature 349:704-706 (1991). Inaddition, all familial forms of autosomal dominant AD have in common anelevated level of the more rapidly aggregating 42 amino acid form of Aβ.Younkin Rinsho Shinkeigaku—Clinical Neurology 37:1099 (1997). Incontrast, no mutation in the tau protein has been shown to cause AD.Instead mutations in tau (chromosome 17) are linked to frontotemporaldementia with Parkinsonism. Goedert et al., Neuron 21:955-958 (1998).Recent evidence has shown a good correlation between the levels of Aβ inbrain and cognitive decline in AD and the deposition of amyloid appearsto be a very early, perhaps the first, event in the pathogenesis of AD,preceding any cognitive impairment. Naslund, et al., JAMA 283:1571-1577(2000). Its presence may modulate a number of biochemical pathways thatresult in the deposition of still other proteins, the activation ofastroglia and microglia, and eventually neuronal cell death andconsequent cognitive dysfunction.

Data suggest that amyloid-binding compounds will have therapeuticpotential in AD and type 2 diabetes mellitus. Morphological reactionsincluding, reactive astrocytosis, dystrophic neurites, activatedmicroglia cells, synapse loss, and full complement activation foundaround neuritic plaques all signify that neurotoxic and celldegenerative processes are occurring in the areas adjacent to these Aβdeposits. Joachim et al., Am. J. Pathol. 135: 309 (1989); Masliah etal., loc. cit. 137: 1293 (1990); Lue and Rogers, Dementia 3: 308 (1992).Aβ-induced neurotoxicity and cell degeneration has been reported in anumber of cell types in vitro. Yankner et al., Science 250: 279 (1990);Roher et al., BBRC 174: 572 (1991); Frautschy et al., Proc. Natl. Acad.Sci. 88: 83362 (1991); Shearman et al., loc. cit. 91: 1470 (1994). Ithas been shown that aggregation of the Aβ peptide is necessary for invitro neurotoxicity. Yankner, Neurobiol. Aging 13: 615 (1992). Recently,three laboratories have reported results which suggest that Congo redinhibits Aβ-induced neurotoxicity and cell degeneration in vitro.Burgevin et al., NeuroReport 5: 2429 (1994); Lorenzo and Yankner, Proc.Natl. Acad. Sci. 91: 12243 (1994); Pollack et al., Neuroscience Letters184: 113 (1995); Pollack et al., Neuroscience Letters 197: 211 (1995).The mechanism appears to involve both inhibition of fibril formation andprevention of the neurotoxic properties of formed fibrils. Lorenzo andYankner, Proc. Natl. Acad. Sci. 91: 12243 (1994). Congo red also hasbeen shown to protect pancreatic islet cells from the toxicity caused byamylin. Lorenzo and Yankner, Proc. Natl. Acad. Sci. 91: 12243 (1994).Amylin is a fibrillar peptide similar to Aβ which accumulates in thepancreas in type 2 diabetes mellitus.

It is known in the art that certain azo dyes, such as Congo red, may becarcinogenic. Morgan et al. Environmental Health Perspectives, 102(supp.) 2: 63-78, (1994). This potential carcinogenicity appears to bebased largely on the fact that azo dyes are extensively metabolized tothe free parent amine by intestinal bacteria. Cemiglia et al., Biochem.Biophys. Res. Com., 107: 1224-1229, (1982). In the case of benzidinedyes (and many other substituted benzidines), it is the free amine whichis the carcinogen. These facts have little implications for amyloidimaging studies in which an extremely minute amount of the high specificactivity radiolabelled dye would be directly injected into the bloodstream. In this case, the amount administered would be negligible andthe dye would by-pass the intestinal bacteria.

In the case of therapeutic usage, these facts have critical importance.Release of a known carcinogen from a therapeutic compound isunacceptable. A second problem with diazo dye metabolism is that much ofthe administered drug is metabolized by intestinal bacteria prior toabsorption. This lowered bioavailability remains a disadvantage even ifthe metabolites released are innocuous.

Thioflavin T is a basic dye first described as a selective amyloid dyein 1959 by Vassar and Culling (Arch. Pathol. 68: 487 (1959)). Schwartzet al. (Zbl. Path. 106: 320 (1964)) first demonstrated the use ofThioflavin S, an acidic dye, as an amyloid dye in 1964. The propertiesof both Thioflavin T and Thioflavin S have since been studied in detail.Kelenyi J. Histochem. Cytochem. 15: 172 (1967); Burns et al. J. Path.Bact. 94:337 (1967); Guntern et al. Experientia 48: 8 (1992); LeVineMeth. Enzymol. 309: 274 (1999). Thioflavin S is commonly used in thepost-mortem study of amyloid deposition in AD brain where it has beenshown to be one of the most sensitive techniques for demonstratingsenile plaques. Vallet et al. Acta Neuropathol. 83: 170 (1992).Thioflavin T has been frequently used as a reagent to study theaggregation of soluble amyloid proteins into beta-sheet fibrils. LeVineProt. Sci. 2: 404 (1993). Quaternary amine derivatives related toThioflavin T have been proposed as amyloid imaging agents, although noevidence of brain uptake of these agents has been presented. Caprathe etal. U.S. Pat. No. 6,001,331.

The inability to assess amyloid deposition in AD until after deathimpedes the study of this devastating illness. A method of quantifyingamyloid deposition before death is needed both as a diagnostic tool inmild or clinically confusing cases as well as in monitoring theeffectiveness of therapies targeted at preventing Aβ deposition.Therefore, it remains of utmost importance to develop a safe andspecific method for diagnosing AD before death by imaging amyloid inbrain parenchyma in vivo. Even though various attempts have been made todiagnose AD in vivo, currently, there are no antemortem probes for brainamyloid. No method has utilized a high affinity probe for amyloid thathas low toxicity, can cross the blood-brain barrier, and binds moreeffectively to AD brain than to normal brain in order to identify ADamyloid deposits in brain before a patient's death. Thus, no in vivomethod for AD diagnosis has been demonstrated to meet these criteria.

To date, the present inventors have developed a series of unchargedderivatives of thioflavin T as amyloid-imaging agents that exhibit highaffinity for amyloid deposits and high permeability across theblood-brain barrier. Extensive in vitro and in vivo studies of theseamyloid-imaging agents represented by BTA-1 suggest that theyspecifically bind to amyloid deposits at concentrations typical of thoseachieved during positron emission tomography studies. In the complexmilieu of human brain, non-specific binding of the amyloid-imagingcompounds is low, even in control brains devoid of amyloid deposits. Atnanomolar concentration, these compounds appear not to bind toneurofibrillary tangles.

The present inventors have determined that varying substitution indifferent positions can increase binding affinity depending uponposition of the substituent.

A need exists for amyloid binding compounds that are non-toxic andbioavailable and, consequently, can be used in therapeutics.

SUMMARY OF THE INVENTION

It is therefore an embodiment of the present invention to providecompounds which allow for a safe and specific method for diagnosing ADbefore death by in vivo imaging of amyloid in brain parenchyma. Thecompounds useful for this purpose are according to the followingformula:

wherein Z is S, NR′, O or C(R′)₂ in which case the tautomeric form ofthe heterocyclic ring may become an indole in which R′ is H or a loweralkyl group:

wherein Y is NR¹R², OR², or SR²;

wherein the nitrogen of

is not a quaternary amine;

wherein each R¹ and R² independently is selected from the groupconsisting of H, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), (C═O)—R′,R_(ph), and (CH₂)_(n)R_(ph) (wherein n=1, 2, 3, or 4 and R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined below for R³-R¹⁰ and R′ is H or a lower alkyl group);

each R³-R¹⁰ independently is selected from the group consisting of H, F,Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3),CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F,Cl, Br or I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′,COOR′, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R¹-R¹⁰ and wherein R′ is H or a lower alkyl group), atri-alkyl tin and a chelating group (with or without a chelated metalgroup) of the form W-L or V-W-L, wherein V is selected from the groupconsisting of —COO—, —CO—, —CH₂O— and —CH₂NH—; W is —(CH₂)_(n) wheren=0, 1, 2, 3, 4, or 5; and L is:

wherein M is selected from the group consisting of Tc and Re.

Another embodiment of the present invention relates to specificcompounds of the above formula which are selected from the groupconsisting of structures 1-45:

It is another embodiment of the present invention to provide an approachfor identifying AD amyloid deposits in brain before a patient's death,using a high-affinity probe for amyloid which has low toxicity, cancross the blood-brain barrier, and can distinguish AD brain from normalbrain. Compounds 1-45 are useful in accomplishing the identifying to ADamyloid deposits.

In a preferred embodiment, at least one of the substituents of thethioflavin compounds 1-45 contain a radiolabel selected from the groupconsisting of ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH₂—CH₂—X*, O—CH₂—CH₂—X*,CH₂—CH₂—CH₂—X*, O— CH₂—CH₂—CH₂—X* (wherein X*=¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br or¹⁸F), ¹⁹F, ¹²⁵I, or a carbon-containing substituent as shown above,e.g., methyl, wherein at least one carbon is ¹¹C or ¹³C.

The present invention also relates to novel compounds having one of thefollowing structures:

In still another embodiment, the amyloid binding compounds 1-45 bind toAβ with a dissociation constant (K_(D)) between 0.0001 and 10.0 μM whenmeasured by binding to synthetic Aβ peptide or Alzheimer's Disease braintissue.

Another embodiment of the invention relates to a method for synthesizingthe amyloid binding compounds of the present invention having at leastone of the substituents selected from the group consisting of ¹³¹I,¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, and ¹⁹F, comprising the step of labelingthe amyloid binding compound wherein at least one of the substituents isa tri-alkyl tin, by reaction of the compound with a ¹³¹I, ¹²⁵I, ¹²³I,⁷⁶Br, ⁷⁵Br, ¹⁸F or ¹⁹F containing substance.

A further embodiment of the present invention relates to apharmaceutical composition for in vivo imaging of amyloid deposits,comprising (a) an amyloid binding compound of structures 1-45 and (b) apharmaceutically acceptable carrier.

In another embodiment of the invention is an in vivo method fordetecting amyloid deposits in a subject, comprising the steps of: (a)administering a detectable quantity of a pharmaceutical compositioncomprising the labeled amyloid binding compound, and detecting thebinding of the compound to amyloid deposit in the subject. In apreferred aspect of this embodiment, the amyloid deposit is located inthe brain of a subject. In a particularly preferred aspect of thisembodiment, the subject is suspected of having a disease or syndromeselected from the group consisting of Alzheimer's Disease, familialAlzheimer's Disease, Down's Syndrome and homozygotes for theapolipoprotein E4 allele. In another particularly preferred aspect ofthis embodiment, the detecting is selected from the group consisting ofgamma imaging, magnetic resonance imaging and magnetic resonancespectroscopy. In a preferred aspect of this embodiment, the gammaimaging is either PET or SPECT. In another preferred aspect of thisembodiment, the pharmaceutical composition is administered byintravenous injection. In another preferred aspect of this embodiment,the ratio of (i) binding of the compound to a brain area other than thecerebellum to (ii) binding of the compound to the cerebellum, in asubject, is compared to the ratio in a normal subject.

Anther embodiment relates to a method of detecting amyloid deposits inbiopsy or post-mortem human or animal tissue comprising the steps of:(a) incubating formalin-fixed or fresh-frozen tissue with a solution ofan amyloid binding compound of the present invention to form a labeleddeposit and then, (b) detecting the labeled deposits. In a preferredaspect of this embodiment, the solution is composed of 25-100% ethanol,with the remainder of the solution being water, wherein the solution issaturated with an amyloid binding compound according to the presentinvention. In a particularly preferred aspect of this embodiment, thesolution is composed of an aqueous buffer (such as tris or phosphate)containing 0-50% ethanol, wherein the solution contains 0.0001 to 100 μMof an amyloid binding compound according to the present invention. In aparticularly preferred aspect of this embodiment, the detecting iseffected by microscopic techniques selected from the group consisting ofbright-field, fluorescence, laser-confocal, and cross-polarizationmicroscopy.

A further embodiment relates to a method of quantifying the amount ofamyloid in biopsy or post-mortem tissue comprising the steps of: a)incubating a radiolabeled derivative of an amyloid binding compound ofstructures 1-45 of the present invention with a homogenate of biopsy orpost-mortem tissue, b) separating the tissue-bound from thetissue-unbound radiolabeled derivative of an amyloid binding compound ofthe present invention, c) quantifying the tissue-bound radiolabeledderivative of an amyloid binding compound of the present invention, andd) converting the units of tissue-bound radiolabeled derivative of anamyloid binding compound of the present invention to units of microgramsof amyloid per 100 mg of tissue by comparison with a standard.

Another embodiment relates to a method of distinguishing an Alzheimer'sdisease brain from a normal brain comprising the steps of: a) obtainingtissue from (i) the cerebellum and (ii) another area of the same brainother than the cerebellum, from normal subjects and from subjectssuspected of having Alzheimer's disease; b) incubating the tissues witha radiolabeled derivative of a thioflavin amyloid binding compound ofstructures 1-45 according to the present invention so that amyloid inthe tissue binds with the radiolabeled derivative of an amyloid bindingcompound of structures 1-45 the present invention; c) quantifying theamount of amyloid bound to the radiolabeled derivative of an amyloidbinding compound of the present invention according to the above recitedmethod; d) calculating the ratio of the amount of amyloid in the area ofthe brain other than the cerebellum to the amount of amyloid in thecerebellum; e) comparing the ratio for amount of amyloid in the tissuefrom normal subjects with ratio for amount of amyloid in tissue fromsubjects suspected of having Alzheimer's disease; and f) determining thepresence of Alzheimer's disease if the ratio from the brain of a subjectsuspected of having Alzheimer's disease is above 90% of the ratiosobtained from the brains of normal subjects.

Another embodiment of the present invention relates to compounds ofstructures 1-45 which are useful in binding specifically to amyloiddeposits over neurofibrillary tangles.

Another embodiment relates to a method of selectively binding to amyloidplaques but not to neurofibrillary tangles in vivo brain tissue whchcontains both by administering an effective amount of a consisting ofone of structures 1-45 so that blood concentration of the administeredcompound remains below 10 nM in vivo.

Yet another embodiment relates to novel compounds of structures 1-45,wherein at least one of the atoms of the formula is replaced with aradiolabel, in particular, wherein said radiolabel is ¹¹C.

Yet another embodiment relates to novel compounds of structures 1-45,wherein at least one of the atoms of the formulae is selected from thegroup consisting of ³H, ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH₂—CH₂—X*,O—CH₂—CH₂—X*, CH₂—CH₂—CH₂—X*, O—CH₂—CH₂—CH₂—X* (wherein X*=¹³¹I, ¹²³I,⁷⁶Br, ⁷⁵Br or ¹⁸F), ¹⁹F, ¹²⁵I, a carbon-containing substituent selectedfrom the group consisting of lower alkyl, (CH₂)_(n)OR′, CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, (C═O)N(R′)₂, O(CO)R′, COOR′, CR′═CR′—R_(ph) andCR₂′—CR₂′—R_(ph) wherein at least one carbon is ¹¹C, ¹³C or ¹⁴C and achelating group (with chelated metal group) of the form W-L* or V-W-L*,wherein V is selected from the group consisting of —COO—, —CO—, —CH₂O—and —CH₂NH—; W is —(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5; and L* is:

wherein M* is ^(99m)Tc.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims. Additionally, alldocuments referred to herein are expressly incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shows the structures of a Thioflavin S and Thioflavin T;

FIG. 2 Shows the structures of two thioflavin derivatives according tothe invention;

FIG. 3 Shows four serial sections of fluorescent dyed brain frontalcortex of an AD patient;

FIG. 4 Shows proposed sites of binding of Chrysamine G and Thioflavin Tin β-sheet fibrils;

FIG. 5 Shows competition assay using Chrysamine G, Thioflavin S andThioflavin T, and derivatives of the present invention (BTA-0, BTA-1 andBTA-2);

FIG. 6 Shows time course radioactivity in the frontal cortex of baboonsinjected with labeled BTA-1, 6-Meo-BTA-1 and 6-Me-BTA-1; and

FIG. 7 Shows a tranverse positron emission tomography image of twolevels of baboon brain following i.v. injection of [N-methyl-¹¹C]BTA-1.

FIG. 8 Shows post-mortem sections of human and transgenic mouse brainstained with a derivative of the present invention (BTA-1).

FIG. 9 Shows in vivo labeling of amyloid plaques and vascular amyloidstained by a derivative of the present invention (BTA-1) in livingtransgenic mice imaged with multiphoton microscopy.

FIG. 10A Shows a data comparison of [³H]binding to homogenates from acontrol brain, Alzheimer's disease brain and non-Alzheimer's diseasedementia brain.

FIG. 10B Shows data ratio from a comparison of the frontal cortex andcerebellum for each individual brain in a control brain, Alzheimer'sdisease brain and non-Alzheimer's disease dementia brain.

FIGS. 11A-F Shows the specificity of the inventive compounds for amyloidplaques over neurofibrillary tangles, where A and B show the entorhinalcortex, C and D show the frontal cortex and E and F show the cerebellumof a Braak stage II control crain.

FIG. 12 Table showing [³H] BTA-1 binding to specified areas of a BraakII Control Brain and a Braak VI AD Brain (AD02).

FIG. 13 Shows a Scratchard plot of the binding of [³H] BTA-1 tohomogenates from AD frontal gray matter and underlying frontal whitematter from the same AD brain.

FIG. 14 Shows an autoradiogram and fluorescent micrograph showing theoverlap of [I-125]6-OH-BTA-0-3′-I binding to plaques and cerebrovascularamyloid and amyloid deposits stained by the amyloid dye, X-34. Left:autoradiogram of [I-125]6-OH-BTA-0-3′-I binding to fresh frozen tissuefrom post-mortem AD brain. The dark areas show the localization of[I-125]6-OH-BTA-0-3′-I and are outlined in red. The structure of[I-125]6-OH-BTA-0-3′-I is shown in the lower left. Right: The same pieceof post-mortem AD brain tissue stained with the amyloid dye, X-34.Bright areas indicate plaques and cerebrovascular amyloid. Center:Overlap of the red outline from the left autoradiogram with the X-34stain showing nearly 1:1 correspondence of [I-125]6-OH-BTA-0-3′-Ibinding to plaques and cerebrovascular amyloid. Inset: Shows a 2.25-foldenlargement of the boxed area in the center figure. Bar represents 1000μm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention exploits the ability of Thioflavin compounds andradiolabeled derivatives thereof to cross the blood brain barrier invivo and bind to Aβ deposited in neuritic (but not diffuse) plaques, toAβ deposited in cerebrovascular amyloid, and to the amyloid consistingof the protein deposited in NFT. The present compounds arenon-quaternary amine derivatives of Thioflavin S and T which are knownto stain amyloid in tissue sections and bind to synthetic AP in vitro.Kelenyi J. Histochem. Cytochem. 15: 172 (1967); Burns et al. J. Path.Bact. 94:337 (1967); Guntern et al. Experientia 48: 8 (1992); LeVineMeth. Enzymol. 309: 274 (1999).

The thioflavin derivatives of the present invention have each of thefollowing characteristics: (1) specific binding to synthetic Aβ in vitroand (2) ability to cross a non-compromised blood brain barrier in vivo.

The method of this invention determines the presence and location ofamyloid deposits in an organ or body area, preferably brain, of apatient. The present method comprises administration of a detectablequantity of a pharmaceutical composition containing an amyloid bindingcompound chosen from structures 1-45, as shown above, called a“detectable compound,” or a pharmaceutically acceptable water-solublesalt thereof, to a patient. A “detectable quantity” means that theamount of the detectable compound that is administered is sufficient toenable detection of binding of the compound to amyloid. An “imagingeffective quantity” means that the amount of the detectable compoundthat is administered is sufficient to enable imaging of binding of thecompound to amyloid.

The invention employs amyloid probes which, in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), or gamma imaging such as positronemission tomography (PET) or single-photon emission computed tomography(SPECT), are used to quantify amyloid deposition in vivo. The term “invivo imaging” refers to any method which permits the detection of alabeled thioflavin derivative which is chosen from structures 1-45, asdescribed above. For gamma imaging, the radiation emitted from the organor area being examined is measured and expressed either as total bindingor as a ratio in which total binding in one tissue is normalized to (forexample, divided by) the total binding in another tissue of the samesubject during the same in vivo imaging procedure. Total binding in vivois defined as the entire signal detected in a tissue by an in vivoimaging technique without the need for correction by a second injectionof an identical quantity of labeled compound along with a large excessof unlabeled, but otherwise chemically identical compound. A “subject”is a mammal, preferably a human, and most preferably a human suspectedof having dementia.

For purposes of in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given label. For instance,radioactive isotopes and ¹⁹F are particularly suitable for in vivoimaging in the methods of the present invention. The type of instrumentused will guide the selection of the radionuclide or stable isotope. Forinstance, the radionuclide chosen must have a type of decay detectableby a given type of instrument. Another consideration relates to thehalf-life of the radionuclide. The half-life should be long enough sothat it is still detectable at the time of maximum uptake by the target,but short enough so that the host does not sustain deleteriousradiation. The radiolabeled compounds of the invention can be detectedusing gamma imaging wherein emitted gamma irradiation of the appropriatewavelength is detected. Methods of gamma imaging include, but are notlimited to, SPECT and PET. Preferably, for SPECT detection, the chosenradiolabel will lack a particulate emission, but will produce a largenumber of photons in a 140-200 keV range. For PET detection, theradiolabel will be a positron-emitting radionuclide such as ¹⁹F whichwill annihilate to form two 511 keV gamma rays which will be detected bythe PET camera.

In the present invention, amyloid binding compounds/probes are madewhich are useful for in vivo imaging and quantification of amyloiddeposition. These compounds are to be used in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), positron emission tomography (PET),and single-photon emission computed tomography (SPECT). In accordancewith this invention, the thioflavin derivatives may be labeled with ¹⁹For ¹³C for MRS/MRI by general organic chemistry techniques known to theart. See, e.g., March, J. ADVANCED ORGANIC CHEMISTRY: REACTIONS,MECHANISMS, AND STRUCTURE (3rd Edition, 1985), the contents of which arehereby incorporated by reference. The thioflavin derivatives also may beradiolabeled with ¹⁸F, ¹¹C, ⁷⁵Br, or ⁷⁶Br for PET by techniques wellknown in the art and are described by Fowler, J. and Wolf, A. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota,J., and Schelbert, H. eds.) 391-450 (Raven Press, NY 1986) the contentsof which are hereby incorporated by reference. The thioflavinderivatives also may be radiolabeled with ¹²³I for SPECT by any ofseveral techniques known to the art. See, e.g., Kulkami, Int. J. Rad.Appl. & Inst. (Part B) 18: 647 (1991), the contents of which are herebyincorporated by reference. In addition, the thioflavin derivatives maybe labeled with any suitable radioactive iodine isotope, such as, butnot limited to ¹³¹I, ¹²⁵I, or ¹²³I, by iodination of a diazotized aminoderivative directly via a diazonium iodide, see Greenbaum, F. Am. J.Pharm. 108: 17 (1936), or by conversion of the unstable diazotized amineto the stable triazene, or by conversion of a non-radioactivehalogenated precursor to a stable tri-alkyl tin derivative which thencan be converted to the iodo compound by several methods well known tothe art. See, Satyamurthy and Barrio J. Org. Chem. 48: 4394 (1983),Goodman et al., J. Org. Chem. 49: 2322 (1984), and Mathis et al., J.Labell. Comp. and Radiopharm. 1994: 905; Chumpradit et al., J. Med.Chem. 34: 877 (1991); Zhuang et al., J. Med. Chem. 37: 1406 (1994);Chumpradit et al., J. Med. Chem. 37: 4245 (1994). For example, a stabletriazene or tri-alkyl tin derivative of thioflavin or its analogues isreacted with a halogenating agent containing ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br,⁷⁵Br, ¹⁸F or ¹⁹F. Thus, the stable tri-alkyl tin derivatives ofthioflavin and its analogues are novel precursors useful for thesynthesis of many of the radiolabeled compounds within the presentinvention. As such, these tri-alkyl tin derivatives are one embodimentof this invention.

The thioflavin derivatives also may be radiolabeled with known metalradiolabels, such as Technetium-99m (^(99m)Tc). Modification of thesubstituents to introduce ligands that bind such metal ions can beeffected without undue experimentation by one of ordinary skill in theradiolabeling art. The metal radiolabeled thioflavin derivative can thenbe used to detect amyloid deposits. Preparing radiolabeled derivativesof Tc^(99m) is well known in the art. See, for example, Zhuang et al.,“Neutral and stereospecific Tc-99m complexes:[99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT)”Nuclear Medicine & Biology 26(2):217-24, (1999); Oya et al., “Small andneutral Tc(v)O BAT, bisaminoethanethiol (N2S2) complexes for developingnew brain imaging agents” Nuclear Medicine & Biology 25(2):135-40,(1998); and Hom et al., “Technetium-99m-labeled receptor-specificsmall-molecule radiopharmaceuticals: recent developments and encouragingresults” Nuclear Medicine & Biology 24(6):485-98, (1997).

The methods of the present invention may use isotopes detectable bynuclear magnetic resonance spectroscopy for purposes of in vivo imagingand spectroscopy. Elements particularly useful in magnetic resonancespectroscopy include ¹⁹F and ¹³C.

Suitable radioisotopes for purposes of this invention includebeta-emitters, gamma-emitters, positron-emitters, and x-ray emitters.These radioisotopes include ¹³¹I, ¹²³I, ¹⁸F, ¹¹C, ⁷⁵Br, and ⁷⁶Br.Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) orSpectroscopy (MRS), according to this invention, include ¹⁹F and ¹³C.Suitable radioisotopes for in vitro quantification of amyloid inhomogenates of biopsy or post-mortem tissue include ¹²⁵I, ¹⁴C, and ³H.The preferred radiolabels are ¹¹C or ¹⁸F for use in PET in vivo imaging,¹²³I for use in SPECT imaging, ¹⁹F for MRS/MRI, and ³H or ¹⁴C for invitro studies. However, any conventional method for visualizingdiagnostic probes can be utilized in accordance with this invention.

The method may be used to diagnose AD in mild or clinically confusingcases. This technique would also allow longitudinal studies of amyloiddeposition in human populations at high risk for amyloid deposition suchas Down's syndrome, familial AD, and homozygotes for the apolipoproteinE4 allele. Corder et al., Science 261: 921 (1993). A method that allowsthe temporal sequence of amyloid deposition to be followed can determineif deposition occurs long before dementia begins or if deposition isunrelated to dementia. This method can be used to monitor theeffectiveness of therapies targeted at preventing amyloid deposition.

Generally, the dosage of the detectably labeled thioflavin derivativewill vary depending on considerations such as age, condition, sex, andextent of disease in the patient, contraindications, if any, concomitanttherapies and other variables, to be adjusted by a physician skilled inthe art. Dosage can vary from 0.001 μg/kg to 10 μg/kg, preferably 0.01μg/kg to 1.0 μg/kg.

Administration to the subject may be local or systemic and accomplishedintravenously, intraarterially, intrathecally (via the spinal fluid) orthe like. Administration may also be intradermal or intracavitary,depending upon the body site under examination. After a sufficient timehas elapsed for the compound to bind with the amyloid, for example 30minutes to 48 hours, the area of the subject under investigation isexamined by routine imaging techniques such as MRS/MRI, SPECT, planarscintillation imaging, PET, and any emerging imaging techniques, aswell. The exact protocol will necessarily vary depending upon factorsspecific to the patient, as noted above, and depending upon the bodysite under examination, method of administration and type of label used;the determination of specific procedures would be routine to the skilledartisan. For brain imaging, preferably, the amount (total or specificbinding) of the bound radioactively labeled thioflavin derivative oranalogue of the present invention is measured and compared (as a ratio)with the amount of labeled thioflavin derivative bound to the cerebellumof the patient. This ratio is then compared to the same ratio inage-matched normal brain.

The pharmaceutical compositions of the present invention areadvantageously administered in the form of injectable compositions, butmay also be formulated into well known drug delivery systems (e.g.,oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous),intracisternal, intravaginal, intraperitoneal, local (powders, ointmentsor drops), or as a buccal or nasal spray). A typical composition forsuch purpose comprises a pharmaceutically acceptable carrier. Forinstance, the composition may contain about 10 mg of human serum albuminand from about 0.5 to 500 micrograms of the labeled thioflavinderivative per milliliter of phosphate buffer containing NaCl. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in REMINGTON'S PHARMACEUTICALSCIENCES, 15th Ed. Easton: Mack Publishing Co. pp. 1405-1412 and1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington:American Pharmaceutical Association (1975), the contents of which arehereby incorporated by reference.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobials, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components of the pharmaceutical composition are adjustedaccording to routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

Particularly preferred pharmaceutical compositions of the presentinvention are those that, in addition to specifically binding amyloid invivo and capable of crossing the blood brain barrier, are also non-toxicat appropriate dosage levels and have a satisfactory duration of effect.

According to the present invention, a pharmaceutical compositioncomprising thioflavin amyloid binding compounds, is administered tosubjects in whom amyloid or amyloid fibril formation are anticipated. Inthe preferred embodiment, such subject is a human and includes, forinstance, those who are at risk of developing cerebral amyloid,including the elderly, nondemented population and patients havingamyloidosis associated diseases and Type 2 diabetes mellitus. The term“preventing” is intended to include the amelioration of celldegeneration and toxicity associated with fibril formation. By“amelioration” is meant the treatment or prevention of more severe formsof cell degeneration and toxicity in patients already manifesting signsof toxicity, such as dementia.

The pharmaceutical composition comprises thioflavin amyloid bindingcompounds described above and a pharmaceutically acceptable carrier. Inone embodiment, such pharmaceutical composition comprises serum albumin,thioflavin amyloid binding compounds and a phosphate buffer containingNaCl. Other pharmaceutically acceptable carriers include aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like, as described, for instance, in REMINGTON'S PHARMACEUTICALSCIENCES, 15th Ed., Easton: Mack Publishing Co., pp. 1405-1412 and1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington:American Pharmaceutical Association (1975), and the UNITED STATESPHARMACOPEIA XVIII. 18th Ed. Washington: American PharmaceuticalAssociation (1995), the contents of which are hereby incorporated byreference.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

According to the invention, the inventive pharmaceutical compositioncould be administered orally, in the form of a liquid or solid, orinjected intravenously or intramuscularly, in the form of a suspensionor solution. By the term “pharmaceutically effective amount” is meant anamount that prevents cell degeneration and toxicity associated withfibril formation. Such amount would necessarily vary depending upon theage, weight and condition of the patient and would be adjusted by thoseof ordinary skill in the art according to well-known protocols. In oneembodiment, a dosage would be between 0.1 and 100 mg/kg per day, ordivided into smaller dosages to be administered two to four times perday. Such a regimen would be continued on a daily basis for the life ofthe patient. Alternatively, the pharmaceutical composition could beadministered intramuscularly in doses of 0.1 to 100 mg/kg every one tosix weeks.

According to the aspect of the invention which relates to a method ofdetecting amyloid deposits in biopsy or post-mortem tissue, the methodinvolves incubating formalin-fixed tissue with a solution of athioflavin amyloid binding compound chosen from structures 1-45,described above. Preferably, the solution is 25-100% ethanol, (with theremainder being water) saturated with a thioflavin amyloid bindingcompound of structures 1-45 according to the invention. Upon incubation,the compound stains or labels the amyloid deposit in the tissue, and thestained or labeled deposit can be detected or visualized by any standardmethod. Such detection means include microscopic techniques such asbright-field, fluorescence, laser-confocal and cross-polarizationmicroscopy.

The method of quantifying the amount of amyloid in biopsy or post-mortemtissue involves incubating a labeled derivative of thioflavin accordingto the present invention, or a water-soluble, non-toxic salt thereof,with homogenate of biopsy or post-mortem tissue. The tissue is obtainedand homogenized by methods well known in the art. The preferred label isa radiolabel, although other labels such as enzymes, chemiluminescentand immunofluorescent compounds are well known to skilled artisans. Thepreferred radiolabel is ¹²⁵I, ¹⁴C or ³H which is contained in asubstituent substituted on one of the compounds of structures 1-45.Tissue containing amyloid deposits will bind to the labeled derivativesof the thioflavin amyloid binding compounds of the present invention.The bound tissue is then separated from the unbound tissue by anymechanism known to the skilled artisan, such as filtering. The boundtissue can then be quantified through any means known to the skilledartisan. The units of tissue-bound radiolabeled thioflavin derivativeare then converted to units of micrograms of amyloid per 100 mg oftissue by comparison to a standard curve generated by incubating knownamounts of amyloid with the radiolabeled thioflavin derivative.

The method of distinguishing an Alzheimer's diseased brain from a normalbrain involves obtaining tissue from (i) the cerebellum and (ii) anotherarea of the same brain, other than the cerebellum, from normal subjectsand from subjects suspected of having Alzheimer's disease. Such tissuesare made into separate homogenates using methods well known to theskilled artisan, and then are incubated with a radiolabeled thioflavinamyloid binding compound. The amount of tissue which binds to theradiolabeled thioflavin amyloid binding compound is then calculated foreach tissue type (e.g. cerebellum, non-cerebellum, normal, abnormal) andthe ratio for the binding of non-cerebellum to cerebellum tissue iscalculated for tissue from normal and for tissue from patients suspectedof having Alzheimer's disease. These ratios are then compared. If theratio from the brain suspected of having Alzheimer's disease is above90% of the ratios obtained from normal brains, the diagnosis ofAlzheimer's disease is made. The normal ratios can be obtained frompreviously obtained data, or alternatively, can be recalculated at thesame time the suspected brain tissue is studied.

The ability of the present compounds to specifically bind toneurofibrially tangles over amyloid plaques is particularly true atconcentrations less than 10 nM, which includes the in vivo concentrationrange of PET radiotraces. At these low concentrations, which containsonly tangles and no plaques, significant binding does not result whencompared to control brain tissue containing neither plaques nor tangles.However, incubation of homogenates of brain tissue which contains mainlyplaques and some tangles with radiolabeled compounds of structures 1-45,results in a significant increase in binding when compared to controltissue without plaques or tangles. This data suggests the advantage thatthese compounds are specific for Aβ deposits at concentrations less than10 nM. These low concentrations are then detectable PET studies, makingPET detection using radiolabeled compounds of structures 1-45 which arespecific for Aβ deposits possible. The use of such compounds permits PETdetection in Aβ deposits such as those found in plaques andcerebrovascular amyloid. Since it has been reported that Aβ levels inthe frontal cortex are increased prior to tangle formation, this wouldsuggest that radiolabeled compounds of structures 1-45, used as PETtracers, would be specific for the earliest changes in AD cortex.Naslund et al. JAMA 283:1571 (2000).

Molecular Modeling

Molecular modeling was done using the computer modeling programAlchemy2000 Tripost, Inc. St. Louis, Mo.) to generate the Aβ peptidechains in the anti-parallel beta-sheet conformation. Kirschner et al.,Proc. Natl. Acad. Sci. U.S.A. 83: 503 (1986). The amyloid peptides wereplaced in hairpin loops (Hilbich et al., J. Mol. Biol. 218: 149 (1991))and used without further structural refinement. The Aβ peptides werealigned so that alternate chains were spaced 4.76 Å apart,characteristic of beta-sheet fibrils. Kirschner, supra. Thioflavin Tderivatives were energy minimized and aligned with the fibril model tomaximize contact with Asp-23/Gln-15/His-13 of Aβ(1-42)

Characterization of Specific Binding to Aβ Synthetic Peptide: Affinity,Kinetics, Maximum Binding

The characteristics of thioflavin derivative binding were analyzed usingsynthetic Aβ(1-40) and 2-(4′-[¹¹C]methylamino-phenyl)-benzothiazole([N-methyl-¹¹C]BTA-1) in phosphate-buffered saline (pH 7.0) or glycinebuffer/20% ethanol (pH 8.0) as previously described for Chysamine-Gbinding. Klunk et al. Neurobiol. Aging 15: 691 (1994).

Amino acid sequence for Aβ(1-40) is as follows: 1 2 3 4 5 6 7 8 9 10 1112 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 13 14 15 16 17 18 1920 21 22 23 24 His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val 25 26 2728 29 30 31 32 33 34 35 36 Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu MetVal 37 38 39 40 Gly Gly Val ValPreparation of Thioflavin Derivatives for Tissue Staining

Both Thioflavin S (ThS) and Thioflavin T (ThT) were utilized aspharmacophores (see, e.g., FIG. 1). It is noted that both compoundscontain quaternary amines and are, therefore, quite hydrophilic as aresult.

[C-14]ThT was synthesized and used to determine relative lipophilicityby partitioning between octanol and phosphate-buffered saline. The logof the partition coefficient, log P_(oct), was found to be 0.57 for[C-14]ThT. It was determined that the quaternary amine renders ThT toopolar for use as an effective brain imaging agent. Based on the resultsof lipophilic Congo red derivatives (phenols uncharged at physiologicpH, but potentially ionizable with a pK_(a) of ˜8.5) (Klunk et al.WO09634853A1, WO09847969A1, WO09924394A2), the inventors removed themethyl group from the benzothiazole nitrogen for the ThT derivatives.The removal of the methyl moiety eliminated the charged quaternary aminefrom the heterocycle portion of the molecule, leaving an aromatic aminewhich typically have pK_(b) values ˜5.5. Shorthand nomenclature for theThT derivatives is used wherein the basic backbone is designated BTA(for BenzoThiazole-Aniline). Substituents on the benzothiazole ring areplaced before the ‘B’ and the number of methyl groups on the anilinenitrogen is placed after the ‘A’ (see, e.g., FIG. 2).

i. Preliminary Tissue Staining with ThT and Derivatives

ThT (see, e.g., FIG. 1) is a fluorescent dye that has been used as ahistological stain for amyloid (Burns et al., “The specificity of thestaining of amyloid deposits with thioflavine T” Journal of Pathology &Bacteriology 94:337-344;1967.). ThT weakly stains plaques (see, e.g.,FIG. 3), tangles, neuropil threads and cerebrovascular amyloid (CVA) inAD brain. Preliminary tissue staining shows that both the primary amine2-(4′-aminophenyl)-6-methyl-benzothiazole (6-Me-BTA-0) and the tertiaryamine 2-(4′-dimethylaminophenyl)-6-methyl-benzothiazole (6-Me-BTA-2)also stain plaques and tangles in post-mortem AD brain (see, e.g., FIG.3). Experiments in which the concentrations of 6-Me-BTA-0 and 6-Me-BTA-2were progressively decreased showed that staining by both 6-Me-BTA-0 and6-Me-BTA-1 could still be detected with staining solutions containingonly 10 nM of the BTA compound. In contrast, BTP (2-phenylbenzothiazole)does not appear to stain plaques, however, this compound is not nearlyas fluorescent as the BTA derivatives. Thus, in the development of thesecompounds, tissue staining has served the dual purpose of assessingspecificity of staining in AD brain tissue as well as assessing bindingaffinity by screening staining solutions over a range of concentrationssimilar to that employed in the binding assays.

ii. Binding Models of Congo Red Derivatives and ThT to Aβ

There are some theories about the binding mechanism of ThT to β-amyloid,but no specific theory has been proven or accepted. However, themechanism appears to be specific and saturable (LeVine, “Quantificationof beta-sheet amyloid fibril structures with thioflavin T” Meth.Enzymol. 309:272-284;1999). Thus, it should be possible to localize thepotential binding site(s) on Aβ and develop a binding model in a manneranalogous to that used to develop the Congo red (CR)/Chrysamine-G (CG)binding model (Klunk et al., “Developments of small molecule probes forthe beta-amyloid protein of Alzheimer's disease” Neurobiol. Aging15:691-698;1994.) based on the following structural and bindingproperties. First, ThT and CG have opposite charges at physiological pH,and it is unlikely that they share a common binding site. This issupported by the lack of competition of ThT for [³H]CG binding to Aβfibrils (see, e.g., FIG. 5).

Previous structural studies of Aβ fibrils (Hilbich et al., “Aggregationand secondary structure of synthetic amyloid beta A4 peptides ofAlzheimer's disease” Journal of Molecular Biology 218:149-63; 1991.) andCR and CG binding to Aβ fibrils suggested a molecular model in which CGbinds through a combination of electrostatic and hydrophobic interactionto the area of Lys-16 (see, e.g., FIG. 4). The studies of LeVine (LeVineibid) help localize the site of ThT binding to Aβ by showing that ThTbinds well to Aβ12-28, but negligibly to Aβ25-35. This suggests the ThTbinding site lies somewhere between residues 12 and 24 of Aβ. It islikely that the positively charged ThT (a quaternary amine) will beattracted to negatively charged (acidic) residues on Aβ. Between aminoacids 12 and 24, the only acidic residues are Glu-22 and Asp-23. Whileboth of these are candidates, the existing model predicts that Glu-22 isinvolved very near the Lys-16 binding site for CG. The current “working”model localizes ThT binding to the area of Asp-23—on the opposite sideof the fibril from the proposed CG site. Since the key feature of ThT(and CG) binding is the presence of a beta-sheet fibrin, binding mustrequire more than just a single amino acid residue. The binding siteexists when residues not normally interacting in monomers are broughttogether in the beta-sheet fibril. Therefore, without being bound to anyone theory, it is believed that ThT also interacts via hydrogen bonds toHis-13 and Gln-15 of a separate, adjacent Aβ molecule comprising thebeta-sheet fibril.

iii. Radiolabeling of ThT and Radioligand Binding Assays

Assessing binding by tissue staining is useful, particularly forassessing specificity. The compound BTP, which is not very fluorescent,may not show staining either because it does not bind well enough, orbecause it is not fluorescent enough. In addition to the AD tissuestaining, quantitative binding assays can be conductedspectrophotometrically (LeVine ibid). This assay depends onmetachromatic spectral shift which occurs when ThT binds to the amyloidfibril. While this assay can be useful to individually screen highlyfluorescent compounds that show this metachromatic shift, it has notbeen determined to be useful for competition assays. For example, it iscommonly observed that test compounds (e.g., CG) quench the fluorescenceof the ThT-Aβ complex (as well as ThT alone). Compounds that quench, butdo not bind to the ThT site, will falsely appear to bind. Therefore, itis preferable to use radiolabeled ThT in typical radioligand bindingassays with aggregated Aβ. In this assay, inhibition of radiolabeled ThTbinding to Aβ trapped on filters would represent true inhibition of ThTbinding and does not require the test compound to be highly fluorescent.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including U.S. patents, are specificallyincorporated into this patent application by reference.

EXAMPLES

All of the reagents used in the synthesis were purchased from AldrichChemical Company and used without further purification. Melting pointswere determined on Mel-TEMP II and were uncorrected. The ¹H NMR spectraof all compounds were measured on Bruker 300 using TMS as internalreference and were in agreement with the assigned structures. The TLCwas performed using Silica Gel 60 F₂₅₄ from EM Sciences and detectedunder UV lamp. Flash chromatography was performed on silica gel 60(230-400 mesh. purchased from Mallinckrodt Company. The reverse phaseTLC were purchased from Whiteman Company.

Synthesis Examples Example 1 Synthesis of2-(4′-aminophenyl)-benzothiazole derivatives

Route 1: Example of the synthesis of 6-MeO-BTA-0, -1, -2, which arerepresentative of the group of thioflavin compounds (Shi et al.,“Antitumor Benzothiazoles. 3. Synthesis of2-(4-Aminophenyl)benzothiazoles and Evaluation of Their Activitiesagainst Breast Cancer Cell Lines in Vitro and in Vivo” J. Med. Chem.39:3375-3384, 1996) (reference numbers of the names compounds belowrefer to the synthetic scheme shown):

(a) 4-Methoxy-4′-nitrobenzanilide (3)

p-Anisidine 1 (1.0 g, 8.1 mmol) was dissolved in anhydrous pyridine (15ml), 4-nitrobenzoyl chloride 2 (1.5 g, 8.1 mmol) was added. The reactionmixture was allowed to stand at room temperature for 16 hrs. Thereaction mixture was poured into water and the precipitate was collectedwith filtrate under vacuum pressure and washed with 5% sodiumbicarbonate (2×10 ml). The product 3 was used in the next step withoutfurther purification. ¹HNMR(300 MHz, DMSO-d₆) δ: 10.46(s, 1H, NH),8.37(d, J=5.5 Hz, 2H, H-3′,5′), 8.17(d, J=6.3 Hz, 2H, H-2′,6′), 7.48(d,J=6.6 Hz, 2H), 6.97(d, J=6.5 Hz, 2H), 3.75(s, 3H, MeO).

(b) 4-Methoxy-4′-nitrothiobenzanilide (4)

A mixture of 4-methoxy-4′-nitrothiobenzaniline 3 (1.0 g, 3.7 mmol) andLawesson's reagent (0.89 g, 2.2 mmol, 0.6 equiv.) in chlorobenzene (15mL) was heated to reflux for 4 hrs. The solvent was evaporated and theresidue was purified with flush column (hexane:ethyl acetate=4:1) togive 820 mg (77.4%) of the product 4 as orange color solid. ¹HNMR(300MHz, DMSO-d₆) δ: 8.29(d, 2H, H-3′,5′), 8.00(d, J=8.5 Hz, 2H, H-2′,6′),7.76(d, 2H), 7.03(d, J=8.4 Hz, 2H), 3.808.37(d, J=5.5 Hz, 2H, H-3′,5′),8.17(d, J=6.3 Hz, 2H, H-2′,6′), 7.48(d, J=6.6 Hz, 2H), 6.97(d, J=6.5 Hz,2H), 3.75(s, 3H, MeO). (s, 3H, MeO).

(c) 6-Methoxy-2-(4-nitrophenyl)benzothiazole (5)

4-Methoxy-4′-nitrothiobenzanilides 4 (0.5 g, 1.74 mmol) was wetted witha little ethanol (0.5 mL), and 30% aqueous sodium hydroxide solution(556 mg 13.9 mmol. 8 equiv.) was added. The mixture was diluted withwater to provide a final solution/suspension of 10% aqueous sodiumhydroxide. Aliquots of this mixture were added at 1 min intervals to astirred solution of potassium ferricyanide (2.29 g, 6.9 mmol, 4 equiv.)in water (5 mL) at 80-90° C. The reaction mixture was heated for afurther 0.5 h and then allowed to cool. The participate was collected byfiltration under vacuum pressure and washed with water, purified withflush column (hexane:ethyl acetate=4:1) to give 130 mg (26%) of theproduct 5.

¹HNMR(300 MHz, Acetone-d₆) δ: 8.45(m, 4H), 8.07(d, J=8.5 Hz, 1H, H-4),7.69(s, 1H, H-7), 7.22(d, J=9.0 Hz, 1H, H-5), 3.90(s, 3H, MeO)

(d) 6-Methoxy-2-(4-aminophenyl)benzothiazole (6)

A mixture of the 6-methoxy-2-(4-nitropheyl)benzothiazoles 5 (22 mg,0.077 mmol) and tin(II) chloride dihydrate (132 mg, 0.45 mmol) inboiling ethanol was stirred under nitrogen for 4 hrs. Ethanol wasevaporated and the residue was dissolved in ethyl acetate (10 mL),washed with 1 N sodium hydroxide (2 mL) and water (5 mL), and dried overMgSO₄. Evaporation of the solvent gave 19 mg (97%) of the product 6 asyellow solid.

(e) 6-Methoxy-2-(4-methylaminophenyl)benzothiazole (7) and6-Methoxy-2-(4-dimethylaminophenyl)benzothiazole (8)

A mixture of 6-methoxy-2-(4-aminophenyl)benzothiazole 6 (15 mg, 0.059mmol), MeI (8.3 mg, 0.060 mmol) and K₂CO₃ (100 mg, 0.72 mmol) in DMSO(anhydrous, 0.5 ml) was heated at 100° C. for 16 hrs. The reactionmixture was purified by reverse phase TLC (MeOH:H₂O=7:1) to give 2.0 mg(13.3%) of 6-methoxy-2-4-methylaminophenylbenzothiazole 7 and 6 mg (40%)of 6-methoxy-2-(4-dimethylaminophenyl)benzothiazole 8. ¹HNMR of 7 (300MHz, Acetone-d₆) δ: 7.85(d, J=8.7 Hz, 2H, H-2′ 6′), 7.75(dd, J=8.8 Hz,J=1.3 Hz, 1H, H-4), 7.49(d, J=2.4 Hz, 1H, H-7), 7.01(dd, J=8.8 Hz, J=2.4Hz, H-5), 6.78(d, J=7.6 Hz, 2H, H-3′ 5′), 3.84(s, 3H, MeO), 2.91(s, 3H,NMe), ¹HNMR of 8 (300 MHz, Acetone-d₆) δ: 7.85(d, J=8.7 Hz, 2H, H-2′6′), 7.75(dd, J=8.8 Hz, J=1.3 Hz, 1H, H-4), 7.49(d, J=2.4 Hz, 1H, H-7),7.01(dd, J=8.8 Hz, J=2.4 Hz, H-5), 6.78(d, J=7.6 Hz, 2H, H-3′ 5′),3.84(s, 3H, MeO), 3.01(s, 6H, NMe₂),

Following the same strategy as above, the other claimed2-(4′-aminophenyl)-benzothiazole derivatives may be synthesized bysubstituting the appropriate substituted aniline derivative (e.g. 2-,3-, or 4-methylaniline) and the appropriate 4-nitro-benzoyl chloridederivative (e.g. 2- or 3-methyl-4-nitro-benzoyl chloride).

Example 2 Synthesis of BTA Derivatives without Substitution

Route 2: Example of the synthesis of BTA-0, -1, -2 compounds, which arerepresentative of the group of thioflavin compounds (Garmaise et al.,“Anthelmintic Quaternary Salts. III. Benzothiazolium Salts” J. Med.Chem. 12:30-36 1969) (reference numbers of the names compounds belowrefer to the synthetic scheme shown):

(a) 2-(4-Nitrophenyl)benzothiazole (19)

A solution of 4-nitrobenzoyl chloride (1.49 g, 8.0 mmol) in benzene(anhydrous, 10 mL) was added dropwise to 2-aminothiophenol (1.0 g, 8.0mmol in 10 ml of benzene) at room temperature. The reaction mixture wasallowed to stir for 16 hr. The reaction was quenched with water (20 mL).The aqueous layer was separated and extracted with ethyl acetate (3×10ml). The combined organic layers were dried and evaporated. The crudeproduct was purified with flush column, (hexane:ethyl acetate=85:15) togive 1.5 g (73.2%) of product as light yellow solid.

(b) 2-(4-Aminophenyl)benzothiazole (20)

A mixture of 2-(4-nitrophenyl)benzothiazole (105 mg, 0.40 mmol) andtin(II) chloride dihydrate (205 mg, 0.91 mmol) in ethanol (20 mL) wasrefluxed under N₂ for 4 hrs. After removing ethanol by vacuumevaporation. The residue was dissolved into ethyl acetate (20 ml), andwashed with NaOH solution (1N, 3×20 ml) and water (3×20 ml), dried andevaporated to dryness to give 102 mg (97%) of the product

(c) 2-(4-Methylaminophenyl)benzothiazole (21) and2-(4-dimethylaminophenyl)benzothiazole (23)

A mixture of 2-(4-aminophenyl)benzothiazole 20 (15 mg, 0.066 mmol), MeI(9.4 mg, 0.066 mg) and K₂CO₃ (135 mg, 0.81 mmol) in DMSO (anhydrous, 0.5ml) was heated at 100° C. for 16 hrs. The reaction mixture was purifiedby reverse phase TLC (MeOH:H₂O=6:1) to give 1.5 mg (10%) of2-(4-methylminophenyl)benzothiazole 21 and 2.5 mg (16.7%) of2-(4-dimethylaminophenyl)benzothiazole 23.

(d) 2-(4-Dimethylaminophenyl)benzothiazole (23)

The mixture of 2-aminothiophenol 9 (0.5 g, 4.0 mmol)4-dimethylaminobenzoic acid 22 (0.66 g, 4.0 mmol) and PPA (10 g) washeated to 220° C. for 4 hrs. The reaction mixture was cooled to roomtemperature and poured into a solution of 10% potassium carbonate (˜400mL). The residue was collected by filtration under vacuum pressure togive 964 mg of the product 23, which was ca. 90% pure based on the ¹HNMRanalysis. Recrystalization of 100 mg of 23 in MeOH gave 80 mg of thepure product.

¹HNMR(300 MHz, Acetone-d₆) δ: 7.12(d, J=7.7 Hz, 1H, H-7), 7.01(d, J=9.0Hz, 1H, H-4), 6.98(d, J=9.1 Hz, 2H, H-2′,6′), 6.56(t, J=7.8 Hz, J=7.3Hz, 1H, H-5 or H-6), 5.92(d, J=8.9 Hz, 1H, H-3′,5′), 2.50(s, 6H, NMe₂).

Following the same strategy as above, the other claimed2-(4′-aminophenyl)-benzothiazole derivatives may be synthesized bysubstituting appropriate 4-nitro-benzoyl chloride derivative (e.g. 2- or3-methyl-4-nitro-benzoyl chloride) or appropriate4-dimethylamino-benzoic acid derivative (e.g. 2- or3-methyl-4-dimethylamino-benzoic acid).

Example 3 Synthesis of Iodinated Compounds

Route 3: Example of the synthesis of2-(3′-Iodo-4′-aminophenyl)-6-hydroxybenzathiazole, which isrepresentative for the synthesis of other iodinated compounds (referencenumbers of the names compounds below refer to the synthetic schemeshown).

(a) 2-(3′-Iodo-4′-aminophenyl)-6-methoxybenzothiazole (24)

To a solution of 2-(4′-aminophenyl)-6-methoxybenzathiazole (22 mg, 0.09mmol) in glacial acetic acid (2.0 mL) was injected 1 M iodochloridesolution in CH₂Cl₂ (0.10 mL, 0.10 mmol, 1.2 eq.) under N₂ atmosphere.The reaction mixture was stirred at room temperature for 16 hr. Theglacial acetic acid was removed under reduced pressure and the residuewas dissolved in CH₂Cl₂. After neutralizing the solution with NaHCO₃,the aqueous layer was separated and extracted with CH₂Cl₂. The organiclayers were combined and dried over MgSO₄. Following the evaporation ofthe solvent, the residue was purified by preparative TLC (Hexanes:ethylacetate=6:1) to give 2-(4′-amino-3′-iodophenyl)-6-methoxybenzathiazole(5) (25 mg, 76%) as brown solid. ¹HNMR (300 MHz, CDCl₃) δ (ppm): 8.35(d, J=2.0 Hz, 1H), 7.87 (dd, J₁=2.0 Hz, J₂=9.0 Hz, 1H), 7.31 (d, J=2.2Hz, 1H), 7.04 (dd, J₁=2.2 Hz, J₂=9.0 Hz, 1H), 6.76 (d, J=9.0 Hz, 1H),3.87 (s, 3H).

(b) 2-(3′-Iodo-4′-aminophenyl)-6-hydroxybenzathiazole (25)

To a solution of 2-(4′-Amino-3′-iodophenyl)-6-methoxybenzathiazole (5)(8.0 mg, 0.02 mmol) in CH₂Cl₂ (2.0 mL) was injected 1 M BBr₃ solution inCH₂Cl₂ (0.20 ml, 0.20 mmol) under N₂ atmosphere. The reaction mixturewas stirred at room temperature for 18 hrs. After the reaction wasquenched with water, the mixture was neutralized with NaHCO₃. Theaqueous layer was extracted with ethyl acetate (3×3 mL). The organiclayers were combined and dried over MgSO₄. The solvent was thenevaporated under reduced pressure and the residue was purified bypreparative TLC (Hexanes:ethyl acetate=7:3) to give2-(3′-iodo-4′-aminophenyl)-6-hydroxybenzothiazole 6 (4.5 mg, 58%) as abrown solid. ¹HNMR (300 MHz, acetone-d₆) δ (ppm): 8.69 (s, 1H), 8.34 (d,J=2.0 Hz, 1H), 7.77 (dd, J₁=2.0 Hz, J₂=8.4 Hz, 1H), 7.76 (d, J=8.8 Hz,1H), 7.40 (d, J=2.4 Hz, 1H), 7.02 (dd, J₁=2.5 Hz, J₂=8.8 Hz, 1H), 6.94(d, J=8.5 Hz, 1H), 5.47 (br., 2H). HRMS m/z 367.9483 (M⁺ calcd forC₁₃H₉N₂OSI 367.9480).

Biological Examples Example 1 Determination of Affinity for Aβ and BrainUptake of Thioflavin Derivatives

Initial competitive binding studies using [³H]CG and synthetic Aβ(1-40)were conducted to determine if CG, ThS and ThT bound to the samesite(s). It has been determined that ThS competed with [³H]CG forbinding sites on AP (1-40), but ThT did not (see, e.g., FIG. 5). Highspecific activity [N-methyl-¹¹C]BTA-1 (see Table 1) was then synthesizedby methylation of BTA-0. Bindings studies were performed with[N-methyl-¹¹C]BTA-1 and 200 nM Aβ(1-40) fibrils. The specific binding of[N-methyl-¹¹C]BTA-1 was ˜70%. FIG. 5 (see the right panel) showscompetition curves for Aβ sites by ThT, BTA-0, BTA-1, and BTA-2 usingthe [N-methyl-¹¹C]BTA-1 binding assay. The Ki's were: 3.0±0.8 nM forBTA-2; 9.6±1.8 nM for BTA-1; 100±16 nM for BTA-0; and 1900±510 nM forThT. Not only is the quaternary amine of ThT not necessary for bindingto Aβ fibrils, it appears to decrease binding affinity as well.

In Table 1 below are five different ¹¹C-labeled BTA derivatives wheretheir in vitro binding properties, log P values, and in vivo brainuptake and retention properties in mice have been determined.

TABLE 1 In vitro and in vivo properties of several promising ¹¹C-labeledThioflavin T derivatives. Ratio of K_(i) Mouse Brain Mouse Brain 2min/30 (nM) Uptake @ 2 Uptake @ 30 min Structure of ¹¹C-Labeled to Aβmin min Uptake BTA Compound fibrils logP (% ID/g*kg) (% ID/g*kg) Values

21 3.3(est.) 0.32 ± 0.07 0.17 ± 0.05 1.9

nottested 3.9(est.) 0.15 ± 0.06 0.16 ± 0.02 0.9

30 1.9(est.) 0.60 ± 0.04 0.39 ± 0.05 1.5

  5.7 2.7 0.43 ± 0.11 0.094 ± 0.038 4.6

  2.3 3.3(est.) 0.32 ± 0.09 0.42 ± 0.10 0.8

  9.6 2.7 0.44 ± 0.14 0.057 ± 0.010 7.7 Structures K_(i) (nM) logP 2 min(% ID/g) 30 min (% ID/g) 2:30 min ratio

8.32 3.17 9.08 3.4 2.7

4.94 3.90 4.40 2.68 1.6

11.1 1.65 5.64 0.36 15.7

3.22 2.35 7.76 2.66 2.91

The data shown in Table 1 indicates that these compounds displayedrelatively high affinity for AP, with Ki values <10 nM, and readilyentered mouse brain with uptake values >0.4%ID/g*kg (or >13% ID/g for 30g animals). Moreover, the 30 min brain radioactivity concentrationvalues were less than 0.1% ID/g*kg, resulting in 2 min-to-30 minconcentration ratios >4. Both of the N,N-dimethyl compounds cleared lessrapidly from mouse brain tissue than the N-methyl derivatives. Likewise,the only primary amine currently testable, 6-MeO-BTA-0, showed poorbrain clearance. This result supports the specific use of the secondaryamine (e.g., —NHCH₃) as in vivo imaging agent.

Example 2 In Vivo PET Imaging Experiments in Baboons

Large amounts of high specific activity (>2000 Ci/mmol) ¹¹C-labeledBTA-1, 6-Me-BTA-1, and 6-MeO-BTA-1 were prepared for brain imagingstudies in 20-30 kg anesthetized baboons using the Siemens/CTI HR+tomograph in 3D data collection mode (nominal FWHM resolution 4.5 mm).Brain imaging studies were conducted following the intravenous injectionof 3-5 mCi of radiotracer. Typical attenuation- and decay-correctedtime-activity curves for a frontal cortex region of interest for each ofthe three compounds are shown in FIG. 6. It is noted that the absolutebrain uptake of these 3 compounds in baboons is very similar to that inmice (i.e., about 0.47 to 0.39% ID/g*kg). However, the normal brainclearance rate of all three radiotracers is considerably slower inbaboons compared to mice, with peak-to-60 min ratios in the range of 2.4to 1.6 compared to ratios as high as 7.7 at 30 min in mice. The rankorder of maximum brain uptake and clearance rate of the three compoundswere also the same in mice and baboons. Brain uptake of the radiotracersdid not appear to be blood flow-limited (FIG. 6, inset). Arterial bloodsamples in the baboons following the injection of all three compoundswere obtained, and showed that their metabolic profiles were quitesimilar. Only highly polar metabolites that eluted near the void volume(4 mL) of the reverse-phase analytical HPLC column were observed in theplasma at all time points following injection, while the unmetabolizedtracer eluted at about 20 mL. Typical amounts of unmetabolized injectatein plasma for all three compounds were about: 90% at 2 minutes; 35% at30 minutes; and 20% at 60 minutes.

Transverse PET images at two levels of baboon brain following the i.v.injection of 3 mCi of [N-methyl-¹¹C]BTA-1 are shown in FIG. 7. Theemission files collected 5-15 min post injection were summed to providethe images. Brain regions include: Ctx (cortex); Thl (thalamus); Occ(occipital cortex); and Cer (cerebellum). FIG. 7 shows the uniformdistribution of radioactivity throughout the brain, indicating lack ofregional binding specificity in normal brain.

Example 3 Staining Amyloid Deposits in Post-Mortem AD and Tg Mouse Brain

Postmortem brain tissue sections from AD brain and an 8 month oldtransgenic PS1/APP mouse were stained with unlabeled BTA-1. The PS1/APPmouse model combines two human gene mutations known to cause Alzheimer'sdisease in a doubly transgenic mouse which deposits Aβ fibrils inamyloid plaques in the brain beginning as early as 3 months of age.Typical fluorescence micrographs are shown in FIG. 8, and the stainingof amyloid plaques by BTA-1 in both postmortem AD and PS1/APP braintissue is clearly visible. Cerebrovascular amyloid also was brightlystained (FIG. 8, right). The other characteristic neuropathologicalhallmark of AD brain, neurofibrillary tangles (NFT), are more faintlystained by BTA-1 in AD brain (FIG. 8, left). NFT have not been observedin transgenic mouse models of amyloid deposition.

Example 4 In Vivo Labeling and Detection of Amyloid Deposits inTransgenic Mice

Three 17 month-old PS1/APP transgenic mice were injectedintraperitoneally (ip) with a single dose of 10 mg/kg of BTA-1 in asolution of DMSO, propylene glycol, and pH 7.5 PBS (v/v/v 10/45/45).Twenty-four hours later, multiphoton fluorescence microscopy wasemployed to obtain high resolution images in the brains of living miceusing a cranial window technique. Typical in vivo images of BTA-1 in aliving PS1/APP mouse are shown in FIG. 9, and plaques andcerebrovascular amyloid are clearly distinguishable. The multiphotonmicroscopy studies demonstrate the in vivo specificity of BTA-1 for Aβin living PS1/APP transgenic mice.

Example 5 Specificity of the Inventive Compounds for Alzheimer Plaquesover Alzheimer Tangles

In order to address the relative contributions of [³H]BTA-1 binding toAβ and tau deposits in the frontal gray of AD brain, [³H]BTA-1 bindingwas compared in homogenates from entorhinal cortex (EC), frontal grayand cerebellum from a typical AD brain and a Braak stage II controlbrain. This control brain had frequent numbers of NFT in the entorhinalcortex (FIG. 5A), but no neuritic or diffuse plaques in any area of thebrain (FIG. 11C). The NFT numbers in the EC of Cntl 04 were similar tothe numbers found in many AD cases (FIG. 11B). [³H]BTA-1 binding in theNFT-rich EC region of this Cntl 04 brain was no greater than [³H]BTA-1binding in the plaque- and NFT-free cerebellum and frontal gray fromthis brain (FIG. 11, Table). A similar survey of these same brain areasin a Braak VI AD brain (FIG. 11, Table), showed low binding incerebellum and EC and over ten-fold higher levels in frontal gray wherethere are frequent numbers of neuritic plaques (FIG. 11D). The extensiveNFT pathology in the EC of the Cntl and AD brains, coupled with the low[3H]BTA-1 binding in the EC suggests that either the BTA-1 binding toNFT seen at 100 nM concentrations of BTA-1 does not occur at 1.2 nM, orthat, at low nanomolar concentrations, the total absolute amount of[³H]BTA-1 binding to NFT deposits is small in comparison to the amountof [³H]BTA-1 bound to Aβ deposits in the plaques and cerebrovascularamyloid of AD frontal gray. The AD brain showed diffuse amyloid plaquedeposits in the EC (FIG. 11B) which did not appear to producesignificant [³H]BTA-1 binding. The frontal cortex had extensive amyloidplaques which were both compact and diffuse and were associated withhigh levels of [³H]BTA-1 binding (FIG. 11D and Table).

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

As used herein and in the following claims, singular articles such as“a”, “an”, and “one” are intended to refer to singular or plural.

1. An amyloid binding compound having a structure selected from thegroup consisting of:


2. An amyloid binding compound having a structure selected from thegroup consisting of:

wherein at least one of the substituents in each of the formulae 1-34,36, and 38-45 is replaced with a substituent selected from the groupconsisting of ³H, ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, ¹⁹F, CH₂—CH₂—X*,O—CH₂—CH₂—X*, CH₂—CH₂—CH₂—X*, O—CH₂—CH₂—CH₂—X*, wherein X*=¹³¹I, ¹²³I,⁷⁶Br, ⁷⁵Br or ¹⁸F, a carbon-containing substituent selected from thegroup consisting of lower alkyl, (CH₂)_(n)OR′, CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X, CN, (C═O)—R′, (C═O)N(R′)₂,O(CO)R′, COOR′, CR′═CR′—R_(ph), and CR₂′—CR₂′—R_(ph), wherein X=F, Cl,Br or I, at least one carbon is ¹¹C, ¹³C or ¹⁴C, R′ is H or a loweralkyl, and R_(ph) is an unsubstituted or substituted phenyl group, withthe phenyl substituents being selected from the group consisting of F,Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3),CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X, CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′ and COOR′), and a chelatinggroup (with chelated metal group) of the form W-L* or V-W-L*, wherein Vis selected from the group consisting of —COO—, —CO—, —CH₂O— and—CH₂NH—, W is —(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5, and L* is:

wherein M* is ^(99m)Tc).
 3. A method of detecting amyloid plaques in apatient in need thereof, comprising (A) administering to said patient adetectable amount of a compound as claimed in claim 2 and (B) detectingthe binding of the compound to amyloid deposits in the patient.
 4. Amethod of synthesizing a compound selected from the group consisting of

wherein at least one of the atoms in each of the formulae 1-34, 36, and38-45 is replaced by a member selected from the group consisting of¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, and ¹⁹F, the method comprisingreacting a tri-alkyl tin derivative of a compound according to one offormulae 1-34, 36, and 38-45 with a halogenating agent containing ¹³¹I,¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, or ¹⁹F.
 5. A pharmaceutical composition forin vivo imaging of amyloid deposits, comprising (a) a compound of claim2 and (b) a pharmaceutically acceptable carrier.
 6. An in vivo methodfor detecting amyloid deposits in a subject, comprising the steps of:(a) administering a detectable quantity of a pharmaceutical compositioncomprising a compound as claimed in claim 2 and (b) detecting thebinding of the compound to amyloid deposits in the subject, wherein thedetecting is selected from the group consisting of gamma imaging,magnetic resonance imaging, and magnetic resonance spectroscopy.
 7. Themethod of claim 6, wherein the amyloid deposit is located in the brainof a subject.
 8. The method of claim 6, wherein the subject is suspectedof having a disease or syndrome selected from the group consisting ofAlzheimer's Disease, familial Alzheimer's Disease, Down's Syndrome andhomozygotes for the apolipoprotein E4 allele.
 9. The method of claim 6,wherein the detecting is done by gamma imaging, and the gamma imaging iseither PET or SPECT.
 10. The method of claim 6, wherein thepharmaceutical composition is administered by intravenous injection. 11.The method of claim 6, wherein the ratio of (i) binding of the compoundto a brain area other than the cerebellum to (ii) binding of thecompound to the cerebellum, in the subject, is compared to the ratio innormal subjects.
 12. A method of detecting amyloid deposits in biopsy orpost-mortem human or animal tissue, comprising the steps of: (a)incubating formalin-fixed or fresh-frozen tissue with a solution of acompound as claimed in claim 2 to form a labeled deposit and then (b)detecting the labeled deposits.
 13. The method of claim 12 wherein thesolution is composed of 25-100% ethanol, with the remainder of thesolution being water, wherein the solution is saturated with thecompound.
 14. The method of claim 12 wherein the solution is composed ofan aqueous buffer containing 0-50% ethanol, wherein the solutioncontains 0.0001 to 100 μM of the compound.
 15. The method of claim 12wherein detecting the labeled deposits is achieved by a microscopictechnique selected from the group consisting of bright-field,fluorescence, laser-confocal, and cross-polarization microscopy.
 16. Amethod of quantifying the amount of amyloid in biopsy or post-mortemtissue comprising the steps of: a) incubating a compound according toone of structures 1-34, 36, and 38-45 with a homogenate of biopsy orpost-mortem tissue, wherein at least one of the substituents in eachstructure is replaced by or substituted with a substituent selected fromthe group consisting of ¹²⁵I, ³H, and a carbon-containing substituent,wherein at least one carbon is ¹⁴C;

b) separating the tissue-bound from the tissue-unbound compound, c)quantifying the tissue-bound compound, and d) converting the units oftissue-bound compound to units of micrograms of amyloid per 100 mg oftissue by comparison with a standard.
 17. A method of distinguishing anAlzheimer's disease brain from a normal brain comprising the steps of:a) obtaining tissue from (i) the cerebellum and (ii) another area of thesame brain other than the cerebellum, from normal subjects and fromsubjects suspected of having Alzheimer's disease; b) incubating thetissues with a compound as claimed in claim 2 so that amyloid in thetissue binds with the compound, then separating the tissue-bound fromthe tissue-unbound compound c) quantifying the amount of amyloid boundto the compound; d) calculating the ratio of the amount of amyloid inthe area of the brain other than the cerebellum to the amount ofaniyloid in the cerebellum; e) comparing the ratio for the amount ofamyloid in the tissue from normal subjects with the ratio for the amountof amyloid in tissue from subjects suspected of having Alzheimer'sdisease; and f) determining the presence of Alzheimer's disease if theratio from the brain of a subject suspected of having Alzheimer'sdisease is above 90% of the ratios obtained from the brains of normalsubjects.
 18. A method of selectively binding a compound to amyloidplaques, but not to neurofibrillary tangles, in brain tissue whichcontains both, comprising contacting the tissue with a compound asclaimed in claim 2 at a concentration which is below 10 nM in in vitrobinding and staining assays.
 19. A method of selectively binding toamyloid plaques but not to neurofibrillary tangles, in in vivo braintissue that contains both, comprising administering to a subject aneffective amount of a compound as claimed in claim 2 so that bloodconcentration of the administered compound remains below 10 mM in vivo.20. A compound according to claim 2, wherein said substituent is ¹¹C.